Anti-cd38 binding domains

ABSTRACT

Provided in this disclosure are anti-CD38 binding domains, a composition comprising the anti-CD38 binding domains, nucleic acids encoding the anti-CD38 binding domains, and a method of using the anti-CD38 binding domains or the composition for treating multiple myeloma.

I. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119 to U.S.Provisional Application No. 62/795,855 filed Jan. 23, 2019, the contentsof which are expressly incorporated by reference in its entirety.

II. REFERENCE TO A “SEQUENCE LISTING”, A TABLE, OR A COMPUTER PROGRAM,LISTING APPENDIX SUBMITTED ON A COMPACT DISK

This invention incorporated by reference the Sequence Listing text copysubmitted herewith, which was created on Jan. 21, 2020, entitled101588-5011-WO_ST25.txt which is 76 kilobytes in size.

III. BACKGROUND OF THE INVENTION

CD38, also known as cyclic ADP ribose hydrolase, is a type IItransmembrane glycoprotein with a long C-terminal extracellular domainand a short N-terminal cytoplasmic domain. CD38 is a member of a groupof related membrane bound or soluble enzymes, comprising CD157 andAplysia ADPR cyclase. This family of enzymes has the unique capacity toconvert NAD to cyclic ADP ribose or nictotinic acid-adenine dinucleotidephosphate.

In addition, CD38 has been reported to be involved in Ca²⁺ mobilizationand in the signal transduction through tyrosine phosphorylation ofnumerous signaling molecules, including phospholipase Cγ, ZAP-70, syk,and c-cbl. Based on these observations, CD38 was proposed to be animportant signaling molecule in the maturation and activation oflymphoid cells during their normal development.

CD38 is found to be expressed on the surface of many immune cells,including B cells, plasma cells, CD4+ T cells, CD8+ T cells, NK cells,NKT cells, mature dendritic cells (DCs) and activated monocytes. Amonghematopoietic cells, CD38 has been found to be involved in functionaleffects such as lymphocyte proliferation, cytokine release, regulationof B and myeloid cell development and survival, and induction ofdendritic cell maturation (FIG. 1).

The presumed natural ligand of CD38 is CD31 (PECAM-1; PlateletEndothelial Cell Adhesion Molecule-1), which is a 130 kD member of theimmunoglobulin superfamily which is expressed on the surface ofcirculating platelets, neutrophils, monocytes, and naïve B-lymphocytes.Functionally, CD31 is thought to act as an adhesion molecule, and theinteraction of CD38 with CD31 may act in promoting survival of leukemiacells.

CD38 is upregulated in many hematopoeitic malignancies and in cell linesderived from various hematopoietic malignancies including non-Hodgkin'slymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), Bchronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia(ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cellleukemia (HCL), Hodgkin's Lymphoma (HL), and chronic myeloid leukemia(CML). CD38 has been hense used as a prognostic marker in leukemia.

In spite of the recent progress in the discovery and development ofanti-cancer agents, many forms of cancer involving CD38-expressingtumors still have a poor prognosis. Thus, there is a need for improvedmethods for treating such forms of cancer, and a need to developanti-CD38 antibodies with improved characteristics despite existingantibodies on the market.

IV. BRIEF SUMMARY OF THE INVENTION

This invention relates to a composition that includes a novel anti-CD38antigen binding domain.

In one aspect, the composition includes an anti-CD38 antigen bindingdomain which contains a variable heavy domain (VH) comprising a vhCDR1with an amino acid sequence of SEQ ID NO:2, a vhCDR2 with an amino acidsequence of SEQ ID NO:3 and a vhCDR3 with an amino acid sequence of SEQID NO:4; and a variable light domain (VL) comprising a vlCDR1 with anamino acid sequence of SEQ ID NO:6, a vlCDR2 with an amino acid sequenceof SEQ ID NO:7 and a vlCDR3 with an amino acid sequence of SEQ ID NO:8.

In some embodiments, said composition includes an anti-CD38 antigenbinding domain that contains a variable heavy domain with an amino acidsequence identical to SEQ ID NO:1, and a variable light domain with anamino acid sequence identical to SEQ ID NO:5. In some embodiments, saidcomposition includes an anti-CD38 antigen binding domain that contains avariable heavy domain with an amino acid sequence identical to SEQ IDNO:1, and a variable light domain with an amino acid sequence identicalto SEQ ID NO:25.

In another aspect, the composition includes an anti-CD38 antigen bindingdomain which contains a variable heavy domain (VH) comprising a vhCDR1with an amino acid sequence of SEQ ID NO:10, a vhCDR2 with an amino acidsequence of SEQ ID NO:11 and a vhCDR3 with an amino acid sequence of SEQID NO:12; and a variable light domain (VL) comprising a vlCDR1 with anamino acid sequence of SEQ ID NO:14, a vlCDR2 with an amino acidsequence of SEQ ID NO:15 and a vlCDR3 with an amino acid sequence of SEQID NO:16.

In some embodiments, said composition includes an anti-CD38 antigenbinding domain that contains a variable heavy domain with an amino acidsequence identical to SEQ ID NO:9, and a variable light domain with anamino acid sequence identical to SEQ ID NO:13.

In another aspect, the composition includes an anti-CD38 antigen bindingdomain which contains a variable heavy domain (VH) comprising a vhCDR1with an amino acid sequence of SEQ ID NO:18, a vhCDR2 with an amino acidsequence of SEQ ID NO:19 and a vhCDR3 with an amino acid sequence of SEQID NO:20; and a variable light domain (VL) comprising a vlCDR1 with anamino acid sequence of SEQ ID NO:22, a vlCDR2 with an amino acidsequence of SEQ ID NO:23 and a vlCDR3 with an amino acid sequence of SEQID NO:24.

In some embodiments, said composition includes an anti-CD38 antigenbinding domain that contains a variable heavy domain with an amino acidsequence identical to SEQ ID NO:17, and a variable light domain with anamino acid sequence identical to SEQ ID NO:21.

In another aspect, the composition includes an anti-CD38 antigen bindingdomain which contains a variable heavy domain (VH) comprising a vhCDR1with an amino acid sequence of SEQ ID NO:54, a vhCDR2 with an amino acidsequence of SEQ ID NO:55 and a vhCDR3 with an amino acid sequence of SEQID NO:56; and a variable light domain (VL) comprising a vlCDR1 with anamino acid sequence of SEQ ID NO:58, a vlCDR2 with an amino acidsequence of SEQ ID NO:59 and a vlCDR3 with an amino acid sequence of SEQID NO:60.

In some embodiments, said composition includes an anti-CD38 antigenbinding domain that contains a variable heavy domain with an amino acidsequence identical to SEQ ID NO:53, and a variable light domain with anamino acid sequence identical to SEQ ID NO:57.

In some embodiments, said composition that includes an anti-CD38 antigenbinding domain as described herein comprises a variable heavy domain anda variable light domain on a single polypeptide. In some embodiments,the single polypeptide includes a scFv linker, a variable heavy domainand a variable light domain in the orientation from N- to C-terminus ofVH-scFv linker-VL or VL-scFv linker-VH.

In some embodiments, said composition that includes an anti-CD38 antigenbinding domain as described herein comprises a first polypeptide whichincludes a variable heavy domain and a second polypeptide which includesa variable light domain.

In some embodiments, said composition that includes an anti-CD38 antigenbinding domain as described herein is antibody that contains a heavychain which includes a variable heavy domain, and a light chain whichincludes a variable light domain. In some embodiments, said antibodycontains a heavy chain which includes a variable heavy domain and aheavy constant domain selected from the heavy constant domains of humanIgG1, IgG2 and IgG4, and the variants thereof. In some embodiments, saidantibody contains a heavy chain which includes a variable heavy domainand a heavy constant domain of human IgG1 or the variants thereof. Insome embodiments, said antibody contains a heavy chain which includes avariable heavy domain and a heavy constant domain selected from theheavy constant domains of human IgG1 variant with ablated FcγR binding.In some embodiments, said antibody contains a heavy chain which includesa variable heavy domain and a heavy constant domain selected from theheavy constant domains of human IgG4 variant with an S228P amino acidsubstitution.

This invention also relates to a nucleic acid composition encoding avariable heavy domain and a variable light domain. In some embodiments,said nucleic acid composition contains a first nucleic acid encoding avariable heavy domain and a second nucleic acid encoding a variableheavy domain. In some embodiments, said nucleic acid compositioncontains a single nucleic acid encoding a variable heavy domain and avariable light domain.

Another aspect of the invention relates to an expression vectorcomposition containing any one of the nucleic acid compositionsdescribed herein; and a host cell containing any one of said expressionvector compositions described herein. In some embodiments, saidexpression vector composition includes a first expression vector thatcontains said first nucleic acid, and a second expression vector thatcontains said second nucleic acid described herein. In some embodiments,said expression vector composition includes said nucleic acidcomposition that contains a single nucleic acid encoding a variableheavy domain and a variable light domain.

This invention further relates to a method of making any of saidcompositions containing an anti-CD38 antigen binding domain describedherein. The method includes culturing said host cell under conditionswherein the anti-CD38 antigen binding domain is expressed, andrecovering said composition.

Also included in this invention is a method of treating multiple myelomausing an effective amount of any of said compositions containing ananti-CD38 antigen binding domain described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the CD38 Expression Profile on Lymphoid Lineage Cells,with a star indicating high CD38 expression. CD38 expression has beenidentified on pro-B cells (CD34+CD19+CD20−), activated B cells(CD19+CD20+), plasma cells (CD138+CD19−CD20−), activated CD4+ and CD8+ Tcells, NKT cells (CD3+CD56+) and NK cells (CD56+CD16+). In addition,CD38 expression is found on lymphoid progenitor cells(CD34+CD45RA+CD10+CD19−) but not the lymphoid stem cell. In addition,increased CD38 expression is seen on mature DCs and activated monocytes.

FIG. 2A-2B show binding of anti-CD38 binding clones to purified humanCD38 by ELISA assay. Each anti-CD38 binding clone was formatted into anscFv and then fused to a Shiga toxin A subunit (scFv-SLTA fusionformat).

FIG. 3A-3B show binding of anti-CD38 binding clones to purifiedcynomolgus CD38 by ELISA assay. Each anti-CD38 binding clone wasformatted into an scFv and then fused to a Shiga toxin A Subunit.

FIG. 4 shows critical contact residues on CD38 extracellular domain foranti-CD38 binding clone CD38TM4 (F216, L262, C119, L124, C201, L230,C254, C275) and anti-CD38 monoclonal antibody daratumumab (S274).

FIG. 5A-5B show binding of anti-CD38 binding clones (in an scFv-SLTAfusion format) to CD38-expressing MOLP-8 cells in the presence ofanti-CD38 antibodies daratumumab (Dara), HB-7, AT13-5 and OKT-10 andseveral fusion proteins. CD38 Targeting reference molecule #1 (CD38TR1)is also in the format of scFv-SLTA fusion protein. FIG. 5C showscompetitive binding of anti-CD38 binding clones including CD38 Targetingreference molecule #1 (CD38TR1) in an scFv-SLTA fusion format toCD38-expressing MOLP-8 cells. All signals were detected by flowcytometry. FIG. 5D is a table summarizing the binding of anti-CD38binding clones in an scFv-SLTA fusion protein format (top row) in thepresence of anti-CD38 binding clones (in scFv alone format) (first leftcolumn).

FIG. 6A-6G depict the sequences of the variable heavy and variable lightdomains of CD38TM1, CD38TM2, CD38TM3, CD38TM4, CD38TM5, CD38TM6 andCD38TR1 as well as the CDRs (underlined in the full domains).

FIG. 7 shows the sequences of the CD38 target antigens of the invention.

FIG. 8 depicts some linker sequences.

FIG. 9 depicts the sequences of some Shiga toxin proteins fused toanti-CD38 binding domains as used in the examples.

FIG. 10A and FIG. 10B depicts a number of IgG heavy constant domain“backbones”, based on human IgG1, IgG2 and IgG4, including some standardvariants, as well as the sequences for the constant light kappa andconstant light lambda sequences. As will be appreciated by those in theart, and as described below, any of these backbones can be combined withthe variable heavy and variable light domains of CD38TM1, CD38TM2,CD38TM3, CD38TM4, CD38TM5, CD38TM6 and CD38TR1.

FIGS. 11A and 11B show purification of CD38TM3-SLTA and CD38TM4-SLTA byIMAC column and protein L (Pro-L) column respectively. MW markerindicates where molecular weight markers are located. FIG. 11C shows thefirst 21 amino acids of CD38TM3 (underlined) is replaced with first 22amino acids of CD38TR1 (underlined) to derive CD38TM4.

FIG. 12 shows illustration of the X-ray structure of a diabody formed bytwo identical scFvs, each containing VH-GGGGS-VL from N- to C-terminus,wherein the VH and VL are from CD38TM4. The VH of one scFv chain (chainA) complexes with the VL of the other scFV chain (chain B) to form aCD38 binding domain.

V DETAILED DESCRIPTION OF THE INVENTION A. Overview

Increased expression of CD38 has been associated with a variety ofdiseases of hematopoietic origin. Such diseases include but are notrestricted to, multiple myeloma, chronic lymphoblastic leukemia, B-cellchronic lymphocytic leukemia, acute lymphoblastic leukemia, includingB-cell acute lymphocytic leukemia, Waldenstrom macroglobulinemia,primary systemic amyloidosis, mantle-cell lymphoma,pro-lymphocytic/myelocytic leukemia, acute myeloid leukemia, chronicmyeloid leukemia, follicular lymphoma, NK-cell leukemia and plasma-cellleukemia. As such, CD38 provides a useful target in the treatment ofdiseases of the hematopoietic system. Several anti-CD38 antibodies arein clinical trials for the treatment of CD38-associated cancers.Accordingly, antibodies to CD38 are useful to treat and diagnose cancer.

The present invention provides anti-CD38 binding domains that are ableto bind human and cynomolgus CD38 with high affinity. Furthermore, theanti-CD38 binding domains disclosed here are capable of binding to CD38in the presence of daratumumab, and hence provide an advantage inclinical applications not seen in some of the existing anti-CD38antibodies in clinical testing.

B. Definitions

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. In someembodiments, it is useful to remove activity from the constant domainsof the antibodies. Thus, for example, “ablating FcγR binding” means theFc region amino acid variant has less than 50% starting binding to anFcγR as compared to an Fc region not containing the specific variant,with less than 70-80-90-95-98% loss of activity being preferred, and ingeneral, with the activity being below the level of detectable bindingin a Biacore assay. For example, one ablation variant in the IgG1constant region is the N297A variant, which removes the nativeglycosylation site and significantly reduces the FcγRIIIa binding andthus reduces the antibody dependent cell-mediated cytotoxicity (ADCC).

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “antigen binding domain” or “ABD” herein is meant a set of sixComplementary Determining Regions (CDRs) that, when present as part of apolypeptide sequence, specifically binds a target antigen as discussedherein. Thus, an “anti-CD38 antigen binding domain” binds CD38 antigenas outlined herein. As is known in the art, these CDRs are generallypresent as a first set of variable heavy CDRs (vhCDRs or VHCDRs) and asecond set of variable light CDRs (vlCDRs or VLCDRs), each comprisingthree CDRs: vhCDR1, vhCDR2, vhCDR3 for the heavy chain and vlCDR1,vlCDR2 and vlCDR3 for the light chain. As is understood in the art, theCDRs are separated by framework regions in each of the heavy variableand light variable regions: for the light variable region, these are(VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4, and for the heavyvariable region, these are(VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4. Antigen bindingdomains of the invention can be embodied in multiple formats, forexample, in Fab, Fv and scFv. In an “Fab” format, the set of 6 CDRs arecontributed by two different polypeptide sequences, the heavy variableregion (vh or VH; containing the vhCDR1, vhCDR2 and vhCDR3) and thelight variable region (vl or VL; containing the vlCDR1, vlCDR2 andvlCDR3), with the C-terminus of the VH being attached to the N-terminusof the CH1 domain of the heavy chain and the C-terminus of the VL beingattached to the N-terminus of the constant light domain (and thusforming the light chain). Heavy variable regions and light variableregions together form Fvs, which can be either scFvs or Fabs, asoutlined herein. Thus, in some cases, the six CDRs of the antigenbinding domain are contributed by a VH and VL. In an scFv format, the VHand VL are covalently attached, generally through the use of a linker asoutlined herein, into a single polypeptide sequence, which can be either(starting from the N-terminus) VH-linker-VL or VL-linker-VH.

By “linker” herein is meant a domain linker that joins two proteindomains together, such as are used in scFv and/or other protein andprotein fusion structures. Generally, there are a number of suitablelinkers that can be used, including traditional peptide bonds, generatedby recombinant techniques that allows for recombinant attachment of thetwo domains with sufficient length and flexibility to allow each domainto retain its biological function. The linker peptide may predominantlyinclude the following amino acid residues: Gly, Ser, Ala, or Thr. Thelinker peptide should have a length that is adequate to link twomolecules in such a way that they assume the correct conformationrelative to one another so that they retain the desired activity. In oneembodiment, the linker is from about 1 to 50 amino acids in length,preferably about 1 to 30 amino acids in length. In one embodiment,linkers of 1 to 20 amino acids in length may be used, with from about 5to about 10 amino acids finding use in some embodiments. Useful linkersinclude glycine-serine polymers, including for example (GS)n, (GSGGS)n,(GGGGS)n, and (GGGS)n, where n is an integer of at least one (andgenerally from 3 to 4), glycine-alanine polymers, alanine-serinepolymers, and other flexible linkers. Alternatively, a variety ofnon-proteinaceous polymers, including but not limited to polyethyleneglycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers ofpolyethylene glycol and polypropylene glycol, may find use as linkers.Other linker sequences may include any sequence of any length of CL/CH1domain but not all residues of CL/CH1 domain; for example, the first5-12 amino acid residues of the CL/CH1 domains. Linkers can also bederived from immunoglobulin light chain, for example Cκ or Cλ. Linkerscan be derived from immunoglobulin heavy chains of any isotype,including for example Cγ1, Cγt, Cγ3, Cγ4, Cα1, Cα2, Cδ, Cε, and Cμ.Linker sequences may also be derived from other proteins such as Ig-likeproteins (e.g., TCR, FcR, KIR), hinge region-derived sequences, andother natural sequences from other proteins. While any suitable linkercan be used, many embodiments utilize a glycine-serine polymer,including for example (GS)n, (GSGGS)n, (GGGGS)n, and (GGGS)n, where n isan integer of at least one (and generally from 2 to 3 to 4 to 5). “scFvlinkers” generally include these glycine-serine polymers, such as thoseshown in FIG. 8. In general, scFv linkers are long enough to allow theVL and VH domains to properly associate to form an antigen bindingdomain (ABD) on a single polypeptide.

The term “antibody” is used in the broadest sense and includes, forexample, an intact immunoglobulin or an antigen binding portion.Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to antibodies or antibody fragments (antibody monomers) thatgenerally are based on the IgG class, which has several subclasses,including, but not limited to IgG1, IgG2, IgG3, and IgG4. In general,IgG1, IgG2 and IgG4 are used more frequently than IgG3. It should benoted that IgG1 has different allotypes with polymorphisms at 356 (D orE) and 358 (L or M). The sequences depicted herein use the 356D/358Mallotype, however the other allotype is included herein. That is, anysequence inclusive of an IgG1 Fc domain included herein can have356E/358L replacing the 356D/358M allotype.

Thus, “isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

As will be appreciated by those in the art, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of avariable heavy and/or variable light sequence includes the disclosure ofthe associated (inherent) CDRs. Accordingly, the disclosure of eachvariable heavy region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2and vhCDR3) and the disclosure of each variable light region is adisclosure of the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

A useful comparison of CDR numbering is as below, see Lafranc et al.,Dev. Comp. Immunol. 27(1):55-77 (2003):

TABLE 1 Kabat + Chothia IMGT Kabat AbM Chothia Contact vhCDR1 26-3527-38 31-35 26-35 26-32 30-35 vhCDR2 50-65 56-65 50-65 50-58 52-56 47-58vhCDR3  95-102 105-117  95-102  95-102  95-102  93-101 vlCDR1 24-3427-38 24-34 24-34 24-34 30-36 vlCDR2 50-56 56-65 50-56 50-56 50-56 46-55vlCDR3 89-97 105-117 89-97 89-97 89-97 89-96

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) and the EU numberingsystem for Fc regions (e.g, Kabat et al., supra (1991)).

The present invention provides a large number of different CDR sets. Inthis case, a “full CDR set” comprises the three variable light and threevariable heavy CDRs, e.g. a vlCDR1, vlCDR2, vlCDR3, vhCDR1, vhCDR2 andvhCDR3. These can be part of a larger variable light or variable heavydomain, respectfully. In addition, as more fully outlined herein, thevariable heavy and variable light domains can be on separate polypeptidechains, when a heavy and light chain is used (for example when Fabs areused), or on a single polypeptide chain in the case of scFv sequences.

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies.

“Epitope” refers to a determinant that interacts with a specific antigenbinding site in the variable region of an antibody molecule known as aparatope. Epitopes are groupings of molecules such as amino acids orsugar side chains and usually have specific structural characteristics,as well as specific charge characteristics. A single antigen may havemore than one epitope. The epitope may comprise amino acid residuesdirectly involved in the binding (also called immunodominant componentof the epitope) and other amino acid residues, which are not directlyinvolved in the binding, such as amino acid residues which areeffectively blocked by the specifically antigen binding peptide; inother words, the amino acid residue is within the footprint of thespecifically antigen binding peptide. Epitopes may be eitherconformational or linear. A conformational epitope is produced byspatially juxtaposed amino acids from different segments of the linearpolypeptide chain. A linear epitope is one produced by adjacent aminoacid residues in a polypeptide chain. Conformational andnon-conformational epitopes may be distinguished in that the binding tothe former but not the latter is lost in the presence of denaturingsolvents.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionN297A refers to a variant polypeptide, in this case an Fc variant, inwhich the asparagine at position 297 is replaced with alanine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, -233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, -233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233#, E233( ) or E233deldesignates a deletion of glutamic acid at position 233. Additionally,EDA233- or EDA233# designates a deletion of the sequence GluAspAla thatbegins at position 233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”. The proteinvariant sequence herein will preferably possess at least about 80%identity with a parent protein sequence, and most preferably at leastabout 90% identity, more preferably at least about 95-98-99% identity.Variant protein can refer to the variant protein itself, compositionscomprising the protein variant, or the DNA sequence that encodes it.

Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification. “Immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,S241P or S228P is a hinge variant with the substitution proline atposition 228 relative to the parent IgG4 hinge polypeptide, wherein thenumbering S228P is according to the EU index and the S241P is the Kabatnumbering. Likewise, M252Y/S254T/T256E defines an Fc variant with thesubstitutions M252Y, S254T and T256E relative to the parent Fcpolypeptide (these mutations increase binding of the Fc domain to theFcRn receptor, thus increasing the half life of the molecule). Theidentity of the wild type amino acid may be unspecified, in which casethe aforementioned variant is referred to as 252Y/254T/256E. It is notedthat the order in which substitutions are provided is arbitrary, that isto say that, for example, 252Y/254T/256E is the same Fc variant as254T/252Y/256E, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to Kabat for the variable regionnumbering and is according to the EU index for the constant regions,including the Fc region. The EU index or EU index as in Kabat or EUnumbering scheme refers to the numbering of the EU antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporatedby reference.) The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand, in some cases, synthetic amino acids. Examples include U.S. Pat.No. 6,586,207; WO 98/48032; WO 03/073238; US2004-0214988A1; WO05/35727A2; WO 05/74524A2; J. W. Chin et al., (2002), Journal of theAmerican Chemical Society 124:9026-9027; J. W. Chin, & P. G. Schultz,(2002), ChemBioChem 11:1135-1137; J. W. Chin, et al., (2002), PICASUnited States of America 99:11020-11024; and, L. Wang, & P. G. Schultz,(2002), Chem. 1-10, all entirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group comprises naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and noreleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1 which incidentally is the position at which the Fc regionis glycosylated. The removal of the glycosylation ablates FcγRIIIabinding, leading to a loss of ADCC activity.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody or antibody fragment.

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.As will be appreciated by those in the art, these are made up of twodomains, a variable heavy domain and a variable light domain.

By “single chain Fv” or “scFv” herein is meant a variable heavy domaincovalently attached to a variable light domain, generally using a scFvlinker as discussed herein, to form a scFv or scFv domain. A scFv domaincan be in either orientation from N- to C-terminus (vh-linker-vl orvl-linker-vh). In general, the linker is a scFv linker as is generallyknown in the art, and discussed above.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification. Similarly, because IgG1 has aproline at position 241 and IgG4 has a serine there, an IgG4 moleculewith a S241P is considered an IgG subclass modification. Note thatsubclass modifications are considered amino acid substitutions herein.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise an asparagine at position 297, the substitutionN297A in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc” or “Fc region” or “Fc domain” as used herein is meant thepolypeptide comprising the constant region of an antibody excluding thefirst constant region immunoglobulin domain (e.g., CH1) and in somecases, part of the hinge. For IgG, the Fc domain comprisesimmunoglobulin domains CH2 and CH3 (Cγ2 and Cγ3) and the lower hingeregion between CH1 (Cγ1) and CH2 (Cγ2). Although the boundaries of theFc region may vary, the human IgG heavy chain Fc region is usuallydefined to include residues C226 or P230 to its carboxyl-terminus,wherein the numbering is according to the EU index as in Kabat.Accordingly, “CH” domains in the context of IgG are as follows: “CH1”refers to positions 118-215 according to the EU index as in Kabat.“Hinge” refers to positions 216-230 according to the EU index as inKabat. “CH2” refers to positions 231-340 according to the EU index as inKabat, and “CH3” refers to positions 341-447 according to the EU indexas in Kabat. Thus, the “Fc domain” includes the —CH2-CH3 domain, andoptionally a hinge domain (hinge-CH2-CH3). In some embodiments, as ismore fully described below, amino acid modifications are made to the Fcregion, for example to alter binding to one or more FcγR receptors or tothe FcRn receptor.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. In some cases, as outlined herein, bindingto one or more of the FcγR receptors is reduced or ablated. For example,reducing binding to FcγRIIIa reduces ADCC, and in some cases, reducingbinding to FcγRIIIa and FcγRIIb is desired.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. As discussedherein, binding to the FcRn receptor is desirable, and in some cases, Fcvariants can be introduced to increase binding to the FcRn receptor.

By “heavy constant region” herein is meant the CH1-hinge-CH2-CH3 portionof a human IgG antibody. A number of suitable heavy constant regions areshown in FIG. 10.

By “light constant region” is meant the CL domain from kappa or lambda,the sequences of which are shown in FIG. 10.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. In the presentcase, the target antigen of interest herein is CD38 protein, usuallyhuman CD38 and optionally cynomolgus CD38, as defined below. Thus, an“anti-CD38 binding domain” is an antigen binding domain (ABD) where theantigen is CD38.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable domain” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ (V.kappa), Vλ (V.lamda), and/or VH genes thatmake up the kappa, lambda, and heavy chain immunoglobulin genetic locirespectively. Thus a “variable heavy domain” comprises(VH)FR1-vhCDR1-(VH)FR2-vhCDR2-(VH)FR3-vhCDR3-(VH)FR4 and a “variablelight domain” comprises(VL)FR1-vlCDR1-(VL)FR2-vlCDR2-(VL)FR3-vlCDR3-(VL)FR4.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities. “Recombinant” means the antibodiesare generated using recombinant nucleic acid techniques in exogeneoushost cells.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

The term “Kassoc” or “Ka”, as used herein, is intended to refer to theassociation rate of a particular antibody-antigen interaction, whereasthe term “Kdis” or “Kd,” as used herein, is intended to refer to thedissociation rate of a particular antibody-antigen interaction. The term“K_(D)”, as used herein, is intended to refer to the dissociationconstant, which is obtained from the ratio of Kd to Ka (i.e., Kd/Ka) andis expressed as a molar concentration (M). K_(D) values for antibodiescan be determined using methods well established in the art. In someembodiments, the method for determining the K_(D) of an antibody is byusing surface plasmon resonance, for example, by using a biosensorsystem such as a BIACORE® system. In some embodiments, the K_(D) of anantibody is determined by Bio-Layer Interferometry. In some embodiments,the K_(D) is measured using flow cytometry with antigen-expressingcells. In some embodiments, the K_(D) value is measured with the antigenimmobilized. In other embodiments, the K_(D) value is measured with theantibody (e.g., parent mouse antibody, chimeric antibody, or humanizedantibody variants) immobilized. In certain embodiments, the K_(D) valueis measured in a bivalent binding mode. In other embodiments, the K_(D)value is measured in a monovalent binding mode. Specific binding for aparticular antigen or an epitope can be exhibited, for example, by anantibody having a K_(D) for an antigen or epitope of at least about 10⁻⁷M, at least about 10⁻⁸ M, at least about 10⁻⁹ M, at least about 10⁻¹⁰ M,at least about 10⁻¹¹ M, at least about 10⁻¹² M, at least about 10⁻¹³ M,at least about 10⁻¹⁴ M. Typically, an antibody that specifically bindsan antigen will have a K_(D) that is 20-, 50-, 100-, 500-, 1000-,5,000-, 10,000- or more times greater for a control molecule relative tothe antigen or epitope.

“Percent (%) amino acid sequence identity” with respect to a proteinsequence is defined as the percentage of amino acid residues in acandidate sequence that are identical with the amino acid residues inthe specific (parental) sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor measuring alignment, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.One particular program is the ALIGN-2 program outlined at paragraphs[0279] to [0280] of US Pub. No. 20160244525, hereby incorporated byreference. Another approximate alignment for nucleic acid sequences isprovided by the local homology algorithm of Smith and Waterman, Advancesin Applied Mathematics, 2:482-489 (1981). This algorithm can be appliedto amino acid sequences by using the scoring matrix developed byDayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5suppl. 3:353-358, National Biomedical Research Foundation, Washington,D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763(1986).

An example of an implementation of this algorithm to determine percentidentity of a sequence is provided by the Genetics Computer Group(Madison, Wis.) in the “BestFit” utility application. The defaultparameters for this method are described in the Wisconsin SequenceAnalysis Package Program Manual, Version 8 (1995) (available fromGenetics Computer Group, Madison, Wis.). Another method of establishingpercent identity in the context of the present invention is to use theMPSRCH package of programs copyrighted by the University of Edinburgh,developed by John F. Collins and Shane S. Sturrok, and distributed byIntelliGenetics, Inc. (Mountain View, Calif.). From this suite ofpackages, the Smith-Waterman algorithm can be employed where defaultparameters are used for the scoring table (for example, gap open penaltyof 12, gap extension penalty of one, and a gap of six). From the datagenerated the “Match” value reflects “sequence identity.” Other suitableprograms for calculating the percent identity or similarity betweensequences are generally known in the art, for example, another alignmentprogram is BLAST, used with default parameters. For example, BLASTN andBLASTP can be used using the following default parameters: geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+Swiss protein+Spupdate+PIR. Details of these programs canbe found at the internet address located by placing http:// in front ofblast.ncbi.nlm.nih.gov/Blast.cgi.

The degree of identity between an amino acid sequence of the presentinvention (“invention sequence”) and the parental amino acid sequence iscalculated as the number of exact matches in an alignment of the twosequences, divided by the length of the “invention sequence,” or thelength of the parental sequence, whichever is the shortest. The resultis expressed in percent identity.

The terms “treatment”, “treating”, “treat”, and the like, refer toobtaining a desired pharmacologic and/or physiologic effect. The effectmay be prophylactic in terms of completely or partially preventing adisease or symptom thereof or reducing the likelihood of a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease. “Treatment”, as used herein, covers any treatment of a diseasein a mammal, particularly in a human, and includes: (a) preventing thedisease from occurring in a subject which may be predisposed to thedisease but has not yet been diagnosed as having it; (b) inhibiting thedisease, i.e., arresting its development or progression; and (c)relieving the disease, i.e., causing regression of the disease and/orrelieving one or more disease symptoms. “Treatment” is also meant toencompass delivery of an agent in order to provide for a pharmacologiceffect, even in the absence of a disease or condition. For example,“treatment” encompasses delivery of a composition that can elicit animmune response or confer immunity in the absence of a diseasecondition, e.g., in the case of a vaccine.

An “effective amount” or “therapeutically effective amount” of acomposition includes that amount of the composition which is sufficientto provide a beneficial effect to the subject to which the compositionis administered. An “effective amount” of a delivery vehicle includesthat amount sufficient to effectively bind or deliver a composition.

The term “nucleic acid” includes RNA or DNA molecules having more thanone nucleotide in any form including single-stranded, double-stranded,oligonucleotide or polynucleotide. The term “nucleotide sequence”includes the ordering of nucleotides in an oligonucleotide orpolynucleotide in a single-stranded form of nucleic acid.

The term “promoter” as used herein includes a DNA sequence operablylinked to a nucleic acid sequence to be transcribed such as a nucleicacid sequence encoding a desired molecule. A promoter is generallypositioned upstream of a nucleic acid sequence to be transcribed andprovides a site for specific binding by RNA polymerase and othertranscription factors.

A “vector” is capable of transferring gene sequences to a target cell.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer a gene sequenceto a target cell, which can be accomplished by genomic integration ofall or a portion of the vector, or transient or inheritable maintenanceof the vector as an extrachromosomal element. Thus, the term includescloning, and expression vehicles, as well as integrating vectors.

The term “regulatory element” as used herein includes a nucleotidesequence which controls some aspect of the expression of a nucleic acidsequence. Examples of regulatory elements illustratively include anenhancer, an internal ribosome entry site (IRES), an intron, an originof replication, a polyadenylation signal (pA), a promoter, an enhancer,a transcription termination sequence, and an upstream regulatory domain,which contribute to the replication, transcription, and/orpost-transcriptional processing of a nucleic acid sequence. In cases,regulatory elements can also include cis-regulatory DNA elements as wellas transposable elements (TEs). Those of ordinary skill in the art arecapable of selecting and using these and other regulatory elements in anexpression construct with no more than routine experimentation.Expression constructs can be generated using a genetic recombinantapproach or synthetically using well-known methodology.

A “control element” or “control sequence” is a nucleotide sequenceinvolved in an interaction of molecules contributing to the functionalregulation of a polynucleotide, including replication, duplication,transcription, splicing, translation, or degradation of thepolynucleotide. The regulation may affect the frequency, speed, orspecificity of the process, and may be enhancing or inhibitory innature. Control elements known in the art include, for example,transcriptional regulatory sequences such as promoters and enhancers. Apromoter is a DNA region capable under certain conditions of binding RNApolymerase and initiating transcription of a coding region usuallylocated downstream (in the 3′ direction) from the promoter.

C. CD38 Protein and Anti-CD38 Binding Domains

1. CD38 Protein

The present invention provides anti-CD38 binding domains thatspecifically bind human and cynomolgus CD38 proteins. The amino acidsequences of human and cynomolgus CD38 proteins are shown in RefSeqaccession identifiers NP_001766.2 (SEQ ID NO:28) and NP_001274206.1 (SEQID NO:30) respectively, with the ECD sequences (SEQ ID NO:29 and SEQ IDNO:31, respectively) shown in FIG. 7.

Accordingly, as used herein, the term “CD38” or “CD38 protein” or “CD38polypeptide” may optionally include any such protein, variants,conjugates, or fragments thereof, including but not limited to known orwildtype CD38, as described herein, as well as any naturally occurringsplice variants, amino acid variants or isoforms, and in particular theextracellular domain (ECD) fragment of CD38. That is, the ABDs of theinvention generally bind to the ECD domains of both human and cyno CD38proteins.

2. Anti-CD38 Binding Domains

The invention provides a number of anti-CD38 binding domains indifferent formats or orientations. Specific CDRs of anti-CD38 bindingdomains are described below. As discussed above, the exact numbering andplacement of the CDRs can be different among different numberingsystems. However, it should be understood that the disclosure of a heavyvariable and/or light variable region includes the disclosure of theassociated (inherent) CDRs. Accordingly, the disclosure of each heavyvariable region is a disclosure of the vhCDRs (e.g. vhCDR1, vhCDR2 andvhCDR3) and the disclosure of each light variable region is a disclosureof the vlCDRs (e.g. vlCDR1, vlCDR2 and vlCDR3).

The present disclosure provides novel anti-CD38 binding domains. Suchantigen binding domains can bind human and cynomolgus CD38 proteins.FIG. 6A-6G depicts the amino acid sequences of four different anti-CD38ABDs that can bind to both human and cynomolgus CD38. In someembodiments, the heavy chain variable region and the light chainvariable region are arranged in a Fab format, which, as discussed below,are optionally included into full length antibodies. In someembodiments, the heavy chain variable region and the light chainvariable region are fused together to form an scFv as generally outlinedherein.

Also included herein are anti-CD38 ABDs that have amino acidmodifications in one or more of the CDRs and/or the framework regions.

As outlined herein, in some embodiments the set of 6 CDRs can have from0, 1, 2, 3, 4, 5 or 6 amino acid modifications (with amino acidsubstitutions finding particular use). That is, the CDRs can have aminoacid modifications (e.g. from 1, 2, 3, 4, 5 or 6 amino acidmodifications in the set of CDRs (that is, the CDRs can be modified aslong as the total number of changes in the set of 6 CDRs is less than 6amino acid modifications, with any combination of CDRs being changed;e.g. there may be one change in vlCDR1, two in vhCDR2, none in vhCDR3,etc.). In some embodiments, amino acid substitutions in the vhCDR3 areless preferred or are avoided. In some cases, the binding affinity foreither or both of human and cyno CD38 may be increased, while in otherembodiments the binding affinity may be reduced. In these embodiments,binding to human and cyno CD38 is retained. Suitable assays for testingwhether an anti-CD38 antigen binding domain that contains modificationsas compared to the VH and VL sequences outlined herein are known in theart, such as Biacore assays or the binding assay outlined in Examples 1,2 or 3.

In some embodiments, the anti-CD38 ABDs outlined herein may also haveamino acid modifications (again, with amino acid substitutions findingparticular use) in the framework regions of either or both of thevariable heavy and variable light framework regions, as long as theframeworks (excluding the CDRs) retain at least about 80, 85 or 90%identity to a human germline sequence. Thus, for example, the identicalCDRs as described herein can be combined with different frameworksequences from human germline sequences, as long as the frameworkregions retain at least 80, 85 or 90% identity to a human germlinesequence.

In another aspect, the invention further provides anti-CD38 bindingdomains that include variants of the above listed heavy chain variableand light chain variable regions. The heavy chain variable regions canbe at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to“VH” sequences herein, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10amino acid changes. The light chain variable regions can be at least 80%(e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to “VL” sequencesherein, and/or contain from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acidchanges. In these embodiments, the invention includes these variants aslong as the antigen binding domains still bind to human and cynomolgusCD38. Suitable assays for testing whether an anti-CD38 antigen bindingdomain that contains mutations as compared to the VH and VL sequencesoutlined herein are known in the art, such as Biacore assays and thoseof Examples 1, 2 and 3.

In some embodiments, the anti-CD38 binding domain is CD38TM1 andincludes a heavy chain variable region having an amino acid sequence atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:9 and a light chain variable region having an amino acid sequenceat least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical toSEQ ID NO:13.

In some embodiments, the anti-CD38 binding domain is CD38TM1 andincludes a vhCDR1 comprising SEQ ID NO:10, a vhCDR2 comprising SEQ IDNO:11, a vhCDR3 comprising SEQ ID NO:12, a vlCDR1 comprising SEQ IDNO:14, a vlCDR2 comprising SEQ ID NO:15, and a vlCDR3 comprising SEQ IDNO:16. In some embodiments, one or more of such 6 CDRs have from 1, 2,3, 4 or 5 amino acid modifications. In further embodiments, any singleCDR contains no more than 1 or 2 amino acid substitutions, and themodified anti-CD38 antigen binding domain retain binding to human and/orcynomolgus CD38.

In some embodiments the anti-CD38 binding domain of CD38TM1 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:9.

In some embodiments the anti-CD38 binding domain of CD38TM1 has a VLdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:13.

In some embodiments the anti-CD38 binding domain of CD38TM1 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:9 and a VL domain that has no more than 1, 2, 3, 4 or 5 amino acidchanges in SEQ ID NO:13.

In some embodiments the anti-CD38 binding domain is CD38TM1 and has a VHwith SEQ ID NO:9 and a VL with SEQ ID NO:13.

In some embodiments, the anti-CD38 binding domain is CD38TM2 andincludes a heavy chain variable region having an amino acid sequence atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:17 and a light chain variable region having an amino acid sequenceat least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical toSEQ ID NO:21.

In some embodiments, the anti-CD38 binding domain is CD38TM2 andincludes a vhCDR1 comprising SEQ ID NO:18, a vhCDR2 comprising SEQ IDNO:19, a vhCDR3 comprising SEQ ID NO:20, a vlCDR1 comprising SEQ IDNO:22, a vlCDR2 comprising SEQ ID NO:23, and a vlCDR3 comprising SEQ IDNO:24. In some embodiments, one or more of such 6 CDRs have from 1, 2,3, 4 or 5 amino acid modifications. In further embodiments, a single CDRcontains no more than 1 or 2 amino acid substitutions, and the modifiedanti-CD38 antigen binding domain retain binding to human and/orcynomolgus CD38.

In some embodiments the anti-CD38 binding domain of CD38TM2 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:17.

In some embodiments the anti-CD38 binding domain of CD38TM1 has a VLdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:21.

In some embodiments the anti-CD38 binding domain of CD38TM2 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:17 and a VL domain that has no more than 1, 2, 3, 4 or 5 aminoacid changes in SEQ ID NO:21.

In some embodiments the anti-CD38 binding domain is CD38TM2 and has a VHwith SEQ ID NO:17 and a VL with SEQ ID NO:21.

In some embodiments, the anti-CD38 binding domain is CD38TM3 andincludes a heavy chain variable region having an amino acid sequence atleast 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQID NO:1 and a light chain variable region having an amino acid sequenceat least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical toSEQ ID NO:5.

In some embodiments, the anti-CD38 binding domain is CD38TM3 andincludes a vhCDR1 comprising SEQ ID NO:2, a vhCDR2 comprising SEQ IDNO:3, a vhCDR3 comprising SEQ ID NO:4, a vlCDR1 comprising SEQ ID NO:6,a vlCDR2 comprising SEQ ID NO:7, and a vlCDR3 comprising SEQ ID NO:8. Insome embodiments, one or more of such 6 CDRs have from 1, 2, 3, 4 or 5amino acid modifications. In further embodiments, a single CDR contains1 or 2 amino acid substitutions, and the modified anti-CD38 antigenbinding domain retain binding to human and/or cynomolgus CD38.

In some embodiments the anti-CD38 binding domain of CD38TM3 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:1.

In some embodiments the anti-CD38 binding domain of CD38TM3 has a VLdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:5.

In some embodiments the anti-CD38 binding domain of CD38TM3 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:1 and a VL domain that has no more than 1, 2, 3, 4 or 5 amino acidchanges in SEQ ID NO:5.

In some embodiments the anti-CD38 binding domain is CD38TM3 and has a VHwith SEQ ID NO:1 and a VL with SEQ ID NO:5.

In some embodiments, the anti-CD38 binding domain is CD38TM4 in thepresent disclosure include a heavy chain variable region having an aminoacid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:1 and a light chain variable region havingan amino acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:25.

In some embodiments, the anti-CD38 binding domain is CD38TM4 andincludes a vhCDR1 comprising SEQ ID NO:2, a vhCDR2 comprising SEQ IDNO:3, a vhCDR3 comprising SEQ ID NO:4, a vlCDR1 comprising SEQ ID NO:6,a vlCDR2 comprising SEQ ID NO:7, and a vlCDR3 comprising SEQ ID NO:8. Insome embodiments, one or more of such 6 CDRs have from 1, 2, 3, 4 or 5amino acid modifications. In further embodiments, a single CDR contains1 or 2 amino acid substitutions, and the modified anti-CD38 antigenbinding domain retain binding to human and/or cynomolgus CD38.

In some embodiments the anti-CD38 binding domain of CD38TM4 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:1.

In some embodiments the anti-CD38 binding domain of CD38TM1 has a VLdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:25.

In some embodiments the anti-CD38 binding domain of CD38TM4 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:1 and a VL domain that has no more than 1, 2, 3, 4 or 5 amino acidchanges in SEQ ID NO:25.

In some embodiments the anti-CD38 binding domain is CD38TM4 and has a VHwith SEQ ID NO:1 and a VL with SEQ ID NO:25.

In some embodiments, the anti-CD38 binding domain is CD38TM5 in thepresent disclosure include a heavy chain variable region having an aminoacid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:53 and a light chain variable region havingan amino acid sequence at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100%) identical to SEQ ID NO:57.

In some embodiments, the anti-CD38 binding domain is CD38TM5 andincludes a vhCDR1 comprising SEQ ID NO:54, a vhCDR2 comprising SEQ IDNO:55, a vhCDR3 comprising SEQ ID NO:56, a vlCDR1 comprising SEQ IDNO:58, a vlCDR2 comprising SEQ ID NO:59, and a vlCDR3 comprising SEQ IDNO:60. In some embodiments, one or more of such 6 CDRs have from 1, 2,3, 4 or 5 amino acid modifications. In further embodiments, a single CDRcontains 1 or 2 amino acid substitutions, and the modified anti-CD38antigen binding domain retain binding to human and/or cynomolgus CD38.

In some embodiments the anti-CD38 binding domain of CD38TM5 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:53.

In some embodiments the anti-CD38 binding domain of CD38TM5 has a VLdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:57.

In some embodiments the anti-CD38 binding domain of CD38TM5 has a VHdomain that has no more than 1, 2, 3, 4 or 5 amino acid changes in SEQID NO:53 and a VL domain that has no more than 1, 2, 3, 4 or 5 aminoacid changes in SEQ ID NO:57.

In some embodiments the anti-CD38 binding domain is CD38TM5 and has a VHwith SEQ ID NO:53 and a VL with SEQ ID NO:57.

In addition to the sequence variants described herein in the heavy chainand light chain variable regions and/or CDRs for each anti-CD38 bindingdomain, changes in the framework region(s) of the heavy and/or lightvariable region(s) can be made. In some embodiments, variations are madein the framework regions that retain at least 80, 85, 90 or 95% identityto the framework region sequences described in Table 1, while keeping 6CDRs unchanged and retaining the binding to human and/or cynomolgusCD38.

In some embodiments, variations are made in both the framework regionsand the 6 CDRs while retaining the binding of the anti-CD38 bindingdomains to human and/or cynomolgus CD38. In these embodiments, the CDRscan have amino acid modifications (e.g., from 1, 2, 3, 4 or 5 amino acidmodifications in the set of 6 CDRs, that is, the CDRs can be modified aslong as the total number of changes in the set of 6 CDRs is less than 6amino acid modifications, with any combination of CDRs being changed;e.g., there may be one change in vlCDR1, two in vhCDR2, none in vhCDR3,etc.).

Epitopes of the anti-CD38 binding domains are mapped as outlined below.The present invention not only includes the enumerated antigen bindingdomains and antibodies herein, but also those that compete for bindingwith the epitopes bound by the enumerated antigen binding domains, e.g.CD38TM1, CD38TM2, CD38TM3, CD38TM4 and CD38TM5. Antigen binding domainsthat recognize the same epitope can be verified in a simple competitiveimmunoassay showing the ability of one antigen binding domain (or anantibody containing such antigen binding domain) to block the binding ofanother antigen binding domain (or an antibody containing such antigenbinding domain) to the target antigen CD38, for example “binning.”Competitive binding studies can be done as is known in the art,generally using SPR/Biacore® binding assays, as well as ELISA andcell-based assays.

D. Characteristics of Anti-CD38 Binding Domains

The present invention provides novel anti-CD38 binding domains. Theanti-CD38 binding domains specifically bind to human and cynomolgusCD38, and preferably the ECD of the human and cynomolgus CD38. Bindingto both human and cynomologous CD38 is useful because cynomolgousanimals may be used as a model for human subjects to help determine,estimate, and understand the in vivo effects and behavior of theCD38-targeting molecule, such as, e.g., regarding pharmacokinetics,pharmacodynamics, and toxicology; wherein the model may have targetbinding and target cell killing by the molecule which is translatable toa related species.

In some embodiments, the anti-CD38 binding domains described herein bindto human and cynomolgus CD38 with high affinities. The K_(D) value canbe measured with the antigen immobilized or with the antibodyimmobilized. The K_(D) value can also be measured in a monovalent or abivalent binding mode. For example, when formatted into IgG1 andmeasured by flow cytometry, the K_(D) values of the antigen bindingdomains binding to human CD38 can be 1×10⁻⁶M or less, 5×10⁻⁷M or less,2.5×10⁻⁷M or less, 1×10⁻⁷M or less, 5×10⁻⁸M or less, 1×10⁻⁸M or less,1×10⁻⁹M or less, or 1×10⁻¹⁰M or less. The K_(D) values of the antigenbinding domains binding to cynomolgus CD38 can be 1×10⁻⁶M or less,5×10⁻⁷M or less, 2.5×10⁻⁷M or less, 1×10⁻⁷M or less, 5×10⁻⁸M or less,2.5×10⁻⁸M or less, 1×10⁻⁸M or less, 5×10⁻⁹M or less, 1×10⁻⁹M or less,5×10⁻¹⁰ M or less, or 1×10⁻¹⁰ M or less. In some embodiments, the K_(D)values of the antigen binding domains binding to human CD38 range from0.1 nM-1 μM, 0.25 nM-500 nM, 0.5 nM-250 nM, 1 nM-100 nM M, or 1.5 nM-50nM. In some embodiments, the K_(D) values of the antigen binding domainsbinding to cynomolgus CD38 range from 0.1 nM-1 μM, 0.25 nM-500 nM, 0.5nM-250 nM, 1 nM-100 nM M, or 2 nM-50 nM.

Furthermore, the anti-CD38 binding domains provided in this disclosurecan bind to various amino acids in the epitope on human CD38 ECD. Insome embodiments, the anti-CD38 binding domains bind to R78 and/or D81.In some embodiments, the anti-CD38 binding domains bind to F216, L262,L124, C201, L230, C254, and/or S274. In some embodiments, the anti-CD38binding domains described herein bind to an epitope on CD38non-overlapping with daratumumab. In some embodiments, the anti-CD38binding domains described herein bind to an epitope on CD38 partiallyoverlapping with daratumumab. In some embodiments, the anti-CD38 bindingdomains described herein are able to bind to human and/or cynomolgusCD38 in the presence of daratumumab.

In some embodiments, one or more of the anti-CD38 binding domainsdisclosed herein are included in a composition, such as in an antibody,which can be used to induce complement-dependent cytotoxicity (CDC) andantibody-dependent cellular cytotoxicity (ADCC) in cells with increasedexpression of CD38, such as multiple myeloma cells.

E. Compositions of Anti-CD38 Binding Domains

As outlined herein, the anti-CD38 binding domains of the invention canbe used in different formats, for example as scFvs or as Fabs includedinto traditional tetrameric antibodies.

1. scFvs Comprising Anti-CD38 Binding Domains

In some embodiments, the composition comprises an scFv that includes ananti-CD38 binding domain described herein. The scFvs binds to human andcynomolgus CD38, and comprises a heavy chain variable region (VH) and alight chain variable region (VL) linked by an scFv linker. The VL and VHcan be in either orientation, e.g. from N- to C-terminus “VH-scFvlinker-VL” or “VL-scFv linker-VH”.

While any suitable linker can be used, many embodiments utilize aglycine-serine polymer, including for example the 15-residue (Gly4Ser)₃peptide (SEQ ID NO:32). Suitable scFv linkers which may be used informing non-covalent multivalent structures include, for example, GGS(SEQ ID NO:33), GGGS (SEQ ID NO:34), GGGGS (SEQ ID NO:35), GGGGSGGG (SEQID NO:36), GGSGGGG (SEQ ID NO:37), as well as GSTSGGGSGGGSGGGGSS (SEQ IDNO:38) or any peptide sequence that allows for recombinant attachment ofthe heavy chain variable region and light chain variable region withsufficient length and flexibility to allow each domain to retain itsbiological function.

Linkers of variable length can be used in this invention. In someembodiments, linkers of 1 to 50 amino acids in length is used. In someembodiments, linkers of 3 to 12 amino acids in length is used, and theresulting scFv monomers tend to form multimers or oligomers (e.g.diabodies, triabodies, and tetrabodies) due to self-association, withthe majority form being dimers. In some embodiments, linkers of 5 aminoacids in length, such as GGGGS (SEQ ID NO:35), is used, and diabodiescan be formed from the ABDs. In some embodiments, linkers of longer than12 (e.g., 13, 14, 15, 16, 17, 18, 19, or 20) amino acids in length isused, and the resulting scFv predominantly forms monomers with only aminority fraction undergoing spontaneous multimerization. In someembodiments, linkers of 1 to 3 amino acids are used, which promotemultimerization of scFv to higher order structures larger than dimericforms, such as trimers and/or mixtures of trimers and tetramers.

Multimerization of scFvs is also included in the present invention. Itmay be controlled by molecular engineering strategies which are eithercovalent or non-covalent, e.g., covalent strategies involvingsingle-chain tandem arrangements, covalent strategies involvingcysteine-mediated, disulfide bond stabilized multimers, and/ornon-covalent strategies involving dimerization domains, linker choice,and/or variable domain order. Multiple strategies (e.g., linker-relatednon-covalent multimerization and covalent disulfide bond stabilization)may be combined to genearate scFv multimers (see e.g. Lu D et al., JImmunol Methods 279: 219-32 (2003)).

In some embodiments, the invention provides ABDs that comprise thevariable heavy and variable light domains, including the VH and VLdomains selected from the group consisting of those of CD38TM1, CD38TM2,CD38TM3, CD38TM4, CD38TM5 and CD38TM6. In some embodiments, the ABDs arein the format of scFv. The VH and VL domains are linked by a 5 aminoacid scFv linker, such as GGGGS (SEQ ID NO:35) to form from N- toC-terminus “VH-scFv linker-VL” or “VL-scFv linker-VH”. Diabodies can beformed from such ABDs. An illustration of the X-ray structure of anexemplary scFv is presented in FIG. 12. This scFv contains a VH and VLfrom CD38TM4 and a scFv linker GGGGS (SEQ ID NO:35), and forms thediabody structure.

2. Antibodies Comprising an Anti-CD38 Binding Domain

In some embodiments, the compositions of the invention are traditional,tetrameric antibodies that comprise the variable heavy and variablelight domains of the invention that form ABDs, including CD38TM1,CD38TM2, CD38TM3, CD38TM4, CD38TM5 and CD38TM6. In general, these VH andVL pairs are added to the heavy chain constant domains(CH1-hinge-CH2-CH3) of human and variant human IgG1, IgG2 and IgG4, thesequences of which are shown in FIG. 10, and the light constant domains(CL) of lambda or kappa, also shown in FIG. 10. In some embodiments, thepresent invention provides a composition comprises an Fab that includesan anti-CD38 binding domain described herein. The C-terminus of theheavy chain variable region (containing the vhCDR1, vhCDR2 and vhCDR3)is attached to the N-terminus of the CH1 domain of the heavy chain, andthe C-terminus of the light chain variable region (containing thevlCDR1, vlCDR2 and vlCDR3) is attached to the N-terminus of the lightchain constant domain. Either the constant lambda or kappa domain can beused in the invention, as well as variants of the CH1 domain and lightchain constant domain described herein.

Suitable heavy chain constant domains include, but are not limited to,those depicted in FIG. 10.

In some embodiments, the antibodies comprise a heavy chain(VH-CH1-hinge-CH2-CH3) with the VH and VL of CD38TM1. In theseembodiments, the constant heavy domain can be from human and variantIgG1, IgG2 and IgG4 as depicted in FIG. 10.

In some embodiments, the VH (SEQ ID NO:9) is made with a heavy constantregion selected from the group consisting of SEQ ID NOs:39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO:13) is made witha constant light domain selected from SEQ ID NOs:51 and 52.

In some embodiments, the antibodies comprise a heavy chain(VH-CH1-hinge-CH2-CH3) with the VH and VL of CD38TM2. In theseembodiments, the constant heavy domain can be from human and variantIgG1, IgG2 and IgG4 as depicted in FIG. 10.

In some embodiments, the VH (SEQ ID NO:17) is made with a heavy constantregion selected from the group consisting of SEQ ID NOs:39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO:21) is made witha constant light domain selected from SEQ ID NOs:51 and 52.

In some embodiments, the antibodies comprise a heavy chain(VH-CH1-hinge-CH2-CH3) with the VH and VL of CD38TM3. In theseembodiments, the constant heavy domain can be from human and variantIgG1, IgG2 and IgG4 as depicted in FIG. 10.

In some embodiments, the VH (SEQ ID NO:1) is made with a heavy constantregion selected from the group consisting of SEQ ID NOs:39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO:5) is made witha constant light domain selected from SEQ ID NOs:51 and 52.

In some embodiments, the antibodies comprise a heavy chain(VH-CH1-hinge-CH2-CH3) with the VH and VL of CD38TM4. In theseembodiments, the constant heavy domain can be from human and variantIgG1, IgG2 and IgG4 as depicted in FIG. 10.

In some embodiments, the VH (SEQ ID NO:1) is made with a heavy constantregion selected from the group consisting of SEQ ID NOs:39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO:25) is made witha constant light domain selected from SEQ ID NOs:51 and 52.

In some embodiments, the antibodies comprise a heavy chain(VH-CH1-hinge-CH2-CH3) with the VH and VL of CD38TM5. In theseembodiments, the constant heavy domain can be from human and variantIgG1, IgG2 and IgG4 as depicted in FIG. 10.

In some embodiments, the VH (SEQ ID NO:53) is made with a heavy constantregion selected from the group consisting of SEQ ID NOs:39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49 or 50, and the VL (SEQ ID NO:57) is made witha constant light domain selected from SEQ ID NOs:51 and 52.

Additional substitutions can be introduced is the Fc region of theantibody as outlined below.

3. Inclusion in Fusion Proteins

In some embodiments, the anti-CD38 binding domains of the invention canbe included in fusion proteins such as generally described in U.S.application Ser. Nos. 14/774,130, 14/965,882, 15/114,487, 15/114,474,15/125,126, 15/125,142, and 15/317,892; and U.S. Provisional ApplicationNos. 62/795,633, 62/945,107 and 62/945,106, such as shown in FIG. 9.

4. Antibody Engineering

The anti-CD38 binding domains and compositions comprising such anti-CD38binding domains can be modified, or engineered, to alter the amino acidsequences by amino acid substitutions. As discussed herein, amino acidsubstitutions can be made to alter the affinity of the CDRs for theprotein (e.g. human and/or cynomolgus CD38, including both increasingand decreasing binding), as well as to alter additional functionalproperties of the antigen binding domains and antibodies.

Additionally, in some embodiments, the Fc regions of the antibodies maybe engineered to include modifications, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fcγ receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antigen binding domain and anantibody according to at least some embodiments of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antigen binding domain and to the antibody) or be modified toalter its glycosylation, or to alter one or more functional propertiesof the antigen binding domain and the antibody. Such embodiments aredescribed further below. The numbering of residues in the Fc region isthat of the EU index of Kabat.

In one embodiment, the hinge region is modified such that the number ofcysteine residues in the hinge region is altered, e.g., increased ordecreased. This approach is described further in U.S. Pat. No. 5,677,425by Bodmer et al. The number of cysteine residues in the hinge region ofCH1 is altered to, for example, facilitate assembly of the light andheavy chains or to increase or decrease the stability of the antibody.

In still another embodiment, the antibody can be modified to abrogate invivo Fab arm exchange, in particular when IgG4 constant domains areused. Specifically, this process involves the exchange of IgG4half-molecules (one heavy chain plus one light chain) between other IgG4antibodies that effectively results in bispecific antibodies which arefunctionally monovalent. Mutations to the hinge region and constantdomains of the heavy chain can abrogate this exchange (see Aalberse, RC, Schuurman J., 2002, Immunology 105:9-19). As outlined herein, amutation that finds particular use in the present invention is the 5241P(Kabat numbering, S228P EU numbering) in the context of an IgG4 constantdomain. IgG4 finds use in the present invention as it has no significanteffector function, and is thus used to block the receptor binding to itsligand without cell depletion.

In some embodiments, amino acid substitutions can be made in the Fcregion, in general for altering binding to FcγR receptors. There are anumber of useful Fc substitutions that can be made to alter binding toone or more of the FcγR receptors. Substitutions that result inincreased binding as well as decreased binding can be useful. Forexample, it is known that increased binding to FcγRIIIa generallyresults in increased ADCC (antibody dependent cell-mediatedcytotoxicity; the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. Similarly, decreasedbinding to FcγRIIb (an inhibitory receptor) can be beneficial as well insome circumstances. Amino acid substitutions that find use in thepresent invention include those listed in U.S. Ser. No. 11/124,620(particularly FIG. 41) and U.S. Pat. No. 6,737,056, both of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor, and/or increase FcRn binding, by modifying one or moreamino acids at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337,338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430,434, 435, 437, 438 or 439. This approach is described further in PCTPublication WO 00/42072 by Presta. Moreover, the binding sites on humanIgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variantswith improved binding have been described (see Shields, R. L. et al.(2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions256, 290, 298, 333, 334 and 339 are shown to improve binding to FcγRIII.Additionally, the following combination mutants are shown to improveFcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224A andS298A/E333A/K334A. Furthermore, mutations such as M252Y/S254T/T256E orM428L/N434S improve binding to FcRn and increase antibody circulationhalf-life (see Chan C A and Carter P J (2010) Nature Rev Immunol10:301-316).

In addition, the antibodies of the invention are modified to increaseits biological half-life. Various approaches are possible. For example,one or more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half-life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al. Additionalmutations to increase serum half-life are disclosed in U.S. Pat. Nos.8,883,973, 6,737,056 and 7,371,826 and include 428L, 434A, 434S, and428L/434S.

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycosylated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for antigen or reduceeffector function such as ADCC. Such carbohydrate modifications can beaccomplished by, for example, altering one or more sites ofglycosylation within the antibody sequence, for example N297. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site, with an alaninereplacement finding use in some embodiments.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies according to at least some embodiments of theinvention to thereby produce an antibody with altered glycosylation. Seefor example, U.S. Patent Publication No. 20040110704 and WO 2003/035835.

Another modification of the antibodies herein that is contemplated bythe invention is PEGylation or the addition of other water solublemoieties, typically polymers, e.g., in order to enhance half-life. Anantibody can be PEGylated to, for example, increase the biological(e.g., serum) half-life of the antibody as is known in the art.

In addition to substitutions made to alter binding affinity to FcγRsand/or FcRn and/or increase in vivo serum half-life, additional antibodymodifications can be made, as described in further detail below.

In some cases, affinity maturation is done. Amino acid modifications inthe CDRs are sometimes referred to as “affinity maturation”. An“affinity matured” antigen binding domain or antibody is one having oneor more alteration(s) in one or more CDRs which results in animprovement in the affinity of the antigen binding domain or antibodyfor antigen, compared to a parent which does not possess thosealteration(s). In some cases, it may be desirable to decrease theaffinity of an antibody to its antigen. Affinity maturation can be doneto increase the binding affinity of the antigen binding domain orantibody for the antigen by at least about 10% to 50-100-150% or more,or from 1 to 5 fold as compared to the “parent” antibody. Preferredaffinity matured antigen binding domains or antibodies will havenanomolar or even picomolar affinities for the antigen. Affinity maturedantibodies are produced by known procedures.

In some embodiments, one or more amino acid modifications are made inone or more of the CDRs of the CD38 binding domains of the invention. Ingeneral, only 1 or 2 or 3-amino acids are substituted in any single CDR,and generally no more than from 1, 2, 3, 4, 5, 6, 7, 8 9 or 10 changesare made within a set of 6 CDRs (e.g. vhCDR1-3 and vlCDR1-3). However,it should be appreciated that any combination of no substitutions, 1, 2or 3 substitutions in any CDR can be independently and optionallycombined with any other substitution.

Alternatively, amino acid modifications can be made in one or more ofthe CDRs of the CD38 binding domains of the invention that are “silent”,e.g. that do not significantly alter the affinity of the antigen bindingdomain or antibody for the antigen. These can be made for a number ofreasons, including optimizing expression (as can be done for the nucleicacids encoding the antigen binding domains or antibodies of theinvention).

F. Nucleic Acids Encoding Antibodies

Nucleic acid compositions encoding the anti-CD38 binding domains andcompositions comprising such antigen binding domains are also provided,as well as expression vectors containing the nucleic acids and hostcells transformed with the nucleic acid and/or expression vectorcompositions. As will be appreciated by those in the art, the proteinsequences depicted herein can be encoded by any number of possiblenucleic acid sequences, due to the degeneracy of the genetic code.

The nucleic acid compositions that encode the compositions comprisinganti-CD38 binding domains will depend on the format of the compositions.Traditional tetrameric antibodies containing two heavy chains and twolight chains are encoded by two different nucleic acids, one encodingthe heavy chain and one encoding the light chain. These can be put intoa single expression vector or two expression vectors, as is known in theart, transformed into host cells, where they are expressed to form theantibodies of the invention. In some embodiments, for example when scFvconstructs are used, a single nucleic acid encoding the heavy variableregion-linker-light variable region is generally used, which can beinserted into an expression vector for transformation into host cells.The nucleic acids can be put into expression vectors that contain theappropriate transcriptional and translational control sequences,including, but not limited to, signal and secretion sequences,regulatory sequences, promoters, origins of replication, selectiongenes, etc.

Preferred mammalian host cells for expressing the recombinant antibodiesaccording to at least some embodiments of the invention include ChineseHamster Ovary (CHO cells), PER.C6, HEK293 and others as is known in theart.

To create a scFv gene, the VH- and VL-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence GGGGS and others discussed herein, suchthat the VH and VL sequences can be expressed as a contiguoussingle-chain protein, with the VL and VH regions joined by the flexiblelinker.

G. Formulations

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the therapeutic function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16^(th) Edition, A. Osal., Ed.,1980). Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed and may includebuffers.

The pharmaceutical composition comprising the anti-CD38 binding domainsor antibodies of the present invention may be formulated for anysuitable route and means of administration, including, but not limitedto intravenous infusion.

For intravenous infusion, in some embodiments, the pharmaceuticalcomposition comprising the anti-CD38 binding domains or antibodies ofthe present invention is formulated in an aqueous buffer solutioncontaining a cryogenic protectant and a surfactant. In some embodiments,the pharmaceutical composition comprising the anti-CD38 binding domainsor antibodies of the present invention is formulated in an aqueoussodium citrate buffer solution containing sucrose as a cryogenicprotectant and polysorbate 80 as a surfactant and is maintained at pH4.8-5.2. In some embodiments, the pharmaceutical composition comprisingthe anti-CD38 binding domains or antibodies of the present invention isformulated in 20 mM sodium citrate buffer, pH 5.0, with 200 mM sucroseand 0.02% volume/volume polysorbate 80. An exemplary formulation islisted below:

Ingredient Amount per mL Function Composition comprising 0.50 mg ActiveIngredient an anti-CD38 binding domain or antibody Sodium Citrate,dihydrate 3.82 mg Buffering agent, conjugated base Citric Acid,monohydrate 1.47 mg Buffering agent, acid Sucrose 68.4 mg Cryogenicprotectant Polysorbate 80  0.2 mg Surfactant stabilizer Water forInjection (WFI) q.s. to 1 mL Solvent Sodium Hydroxide As needed toadjust to Base, adjust pH pH 4.8 to 5.2 Hydrochloric Acid As needed toadjust to Acid, adjust pH pH 4.8 to 5.2

The formulations of the pharmaceutical compositions of the invention mayconveniently be presented in unit dosage form and may be prepared by anyof the methods well known in the art of pharmacy. In such form, thecomposition is divided into unit doses containing appropriate quantitiesof the active component.

The dosing amounts and frequencies of administration are, in a preferredembodiment, selected to be therapeutically or prophylacticallyeffective. As is known in the art, adjustments for protein degradation,systemic versus localized delivery, and rate of new protease synthesis,as well as the age, body weight, general health, sex, diet, time ofadministration, drug interaction and the severity of the condition maybe necessary, and will be ascertainable with routine experimentation bythose skilled in the art.

H. Methods for Using Antibodies

The anti-CD38 binding domains and composition comprising such anti-CD38binding domains of the invention can be used in a number of diagnosticand therapeutic applications.

1. Diagnostic Uses

The anti-CD38 binding domains and composition comprising such anti-CD38binding domains of the invention can find use in the in vitro or in vivodiagnosis of CD38-expressing cancers, including imaging of tumors thatoverexpress CD38.

Generally, diagnosis can be done in several ways. In one embodiment, atissue from a patient, such as a biopsy sample, is contacted with ananti-CD38 antibody, generally labeled, such that the antibody binds tothe endogenous CD38. The level of signal from the label is compared tothat of normal non-cancerous tissue either from the same patient or areference sample, to determine the presence or absence ofCD38-expressing cancer. The biopsy sample can be from a solid tumor, ablood sample (for lymphomas and leukemias such as multiple myeloma, ALL,T cell lymphoma, etc). In general, in this embodiment, the anti-CD38antibody is labeled, for example with a fluorophore or other opticallabel, that is detected using a fluorometer or other optical detectionsystem as is well known in the art.

In another embodiment, a labeled secondary antibody is contacted withthe sample, for example using an anti-human IgG antibody from adifferent mammal (mouse, rat, rabbit, goat, etc.) to form a sandwichassay as is known in the art. Alternatively, the anti-CD38 antibodycould be directly labeled (i.e. biotin) and detection can be done by asecondary antibody directed to the labeling agent in the art.

Once overexpression of CD38 is seen, treatment can proceed with theadministration of an anti-CD38 antigen binding domain or a compositioncomprising an anti-CD38 binding domain according to the invention asoutlined herein.

In other embodiments, in vivo diagnosis is done. Generally, in thisembodiment, the anti-CD38 antibody (including antibody fragments) isinjected into the patient and imaging is done. In this embodiment, forexample, the antibody is generally labeled with an optical label or anMRI label, such as a gadolinium chelate, radioactive labeling of mAb(including fragments).

Particularly useful antibodies for use in diagnosis include, but are notlimited to the enumerated antibodies, or antibodies that utilize theCDRs with variant sequences, or those that compete for binding with anyof the antibodies described herein.

In many embodiments, a diagnostic antibody is labeled. By “labeled”herein is meant that the antibodies disclosed herein have one or moreelements, isotopes, or chemical compounds attached to enable thedetection in a screen or diagnostic procedure. In general, labels fallinto several classes: a) immune labels, which may be an epitopeincorporated as a fusion partner that is recognized by an antibody, b)isotopic labels, which may be radioactive or heavy isotopes, c) smallmolecule labels, which may include fluorescent and colorimetric dyes, ormolecules such as biotin that enable other labeling methods, and d)labels such as particles (including bubbles for ultrasound labeling) orparamagnetic labels that allow body imagining. Labels may beincorporated into the antibodies at any position and may be incorporatedin vitro or in vivo during protein expression, as is known in the art.

Diagnosis can be done either in vivo, by administration of a diagnosticantibody that allows whole body imaging as described below, or in vitro,on samples removed from a patient. “Sample” in this context includes anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, urine, serum, lymph, saliva, analand vaginal secretions, perspiration and semen), as well as tissuesamples such as result from biopsies of relevant tissues.

In addition, as outlined below, information regarding the proteinexpression levels of CD38 can be used to determine which antibodiesshould be administered to a patient.

2. Disease Treatment

The anti-CD38 binding domains and composition comprising such anti-CD38binding domains of the invention find particular use in treatingdiseases, disorders, and/or conditions which may be mediated, regulatedor otherwise affected by CD38 overexpression in a subject or a humanpatient in need thereof. The treatment comprises administering to thesubject or patient a therapeutically effective amount of a compositioncomprising an anti-CD38 binding domain of the invention. Among certainembodiments of the present invention is a composition comprising ananti-CD38 binding domains of the invention for the treatment orprevention of a cancer or immune disorder associated with CD38overexpression. In some embodiments, the disease, disorder, or conditionto be treated using this method of the invention is hematopoieticcancers containing CD38-overpressing cells such as multiple myeloma, andmore generally, in conditions associated with loss of growth control inCD38 expressing cells. Apart from multiple myeloma, CD38 is alsoupregulated in many other hematopoietic malignancies and in cell linesderived from various hematopoietic malignancies, such as non-Hodgkin'slymphoma (NHL), Burkitt's lymphoma (BL), B chronic lymphocytic leukemia(B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma(TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL),Hodgkin's Lymphoma (HL), and chronic myeloid leukemia (CML).

In some embodiments, the anti-CD38 binding domains and/or compositioncomprising such anti-CD38 binding domains of the invention are used incombination with an existing anti-CD38 antibody, e.g., daratumumab, forthe treatment of a cancer or immune disorder associated with CD38overexpression, such as multiple myeloma. In some embodiments, theanti-CD38 binding domains and/or composition comprising such anti-CD38binding domains of the invention are used to treat patient associatedwith CD38 overexpression, such as a multiple myeloma patient, who hasalready been exposed to an anti-CD38 antibody, such as, e.g.,daratumumab. In some embodiments, the anti-CD38 binding domains and/orcomposition comprising such anti-CD38 binding domains of the inventionare used to treat patient associated with CD38 overexpression, such as amultiple myeloma patient, who has suffered failed treatment by anexisting anti-CD38 antibody, e.g., daratumumab. In some embodiments, theanti-CD38 binding domains and/or composition comprising such anti-CD38binding domains are used in combination with an existing anti-CD38antibody for the treatment of diseases associated with CD38overexpression, such as multiple myeloma, wherein the anti-CD38 bindingdomains bind to an epitope on CD38 different from the epitope bound bythe existing anti-CD38 antibody.

VI. EXAMPLES

The invention now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and is not intended to limit the invention.

A. Example 1: Anti-CD38 Antigen Binding Clones Bind to Human andCynomolgus CD38

1. Anti-CD38 Antigen Binding Clones Bind to Cells Expressing CD38

Anti-CD38 antigen binding clones were formatted into human IgG1 andtheir binding to CD38 protein was assayed. Human multiple myeloma cellsMOLP-8 expressing human CD38 and cells expressing recombinant cynomolgusCD38 were incubated with an anti-CD38 antibody or a secondaryantibody-only control at a 100 nM concentration. Antibody binding toCD38 was detected using a fluorophore conjugated anti-human IgGsecondary antibody. Cells were then analyzed by flow cytometry, andfold-over-background (FOB) was calculated using the mean fluorescenceintensity (MFI) of anti-CD38 antibody in comparison with the secondaryantibody only control. Anti-CD38 binding clones bind to both human andcynomolgus CD38, and exemplary data from clones CD38TM3, CD38TM1 andCD38TM2, CD38TM5 and CD38TM6 is shown in Table 2.

TABLE 2 Clone Human CD38 (FOB) Cynomolgus CD38 (FOB) CD38TM3 212.2 528.2CD38TM1 397.1 1920.1 CD38TM2 39.6 271.6 CD38TM5 550.1 104.5 CD38TM6104.1 176.1

2. Anti-CD38 Antigen Binding Clones Bind to Purified CD38

Binding of anti-CD38 antigen binding clones to purified human andcynomolgus CD38 molecules was also tested. Anti-CD38 antigen bindingclones were formatted into scFv, which were then fused to a Shiga toxinA subunit to generate CD38-targeting molecules. For each anti-CD38 scFv,heavy chain variable region (VH) was either fused to the N terminus or Cterminus of the light chain variable region (VL) via the GGGGS linker. Areference anti-CD38 binding clone fused with Shiga toxin A subunit (CD38Targeting Reference molecule #1-SLTA, or CD38TR1-SLTA) was also includedin the test, as well as a modified anti-CD38 binding clone CD38TM4 fusedto Shiga toxin A subunit, which contains amino acid substitutions in thelight chain framework region of CD38TM3 to enable purification byprotein L chromatography. Amino acid sequences of anti-CD38/Shiga toxinfusion protein tested are: SEQ ID NO:40 (CD38TM1-SLTA), SEQ ID NO:41(CD38TM2-SLTA), SEQ ID NO:42 (CD38TM3-SLTA), SEQ ID NO:43 (CD38TM4-SLTA)and SEQ ID NO:44 (CD38TR1-SLTA).

Briefly, Nunc maxisorp plates were coated with recombinant human orcynomolgus monkey CD38 in PBS overnight at 4° C. The wells were washedand blocked, and then incubated with a serial dilution of theCD38-targeting molecules. CD38-targeting molecules bound to the wellswere detected by a two-step method including a murine monoclonalantibody which detects the Shiga toxin domain, and then an anti-mousehorseradish peroxidase (HRP) conjugated secondary antibody. HRP activitywas detected by Ultra TMB ELISA Substrate (Thermo Fisher) and theabsorbance was read at 450 nM.

Anti-CD38 antigen binding clones CD38TM1, CD38TM2, CD38TM3 and CD38TM4bind to both human and cynomolgus CD38. In contrast, CD38TR1 only bindsto human CD38. Binding of each anti-CD38 binding clone was tested in theformat of an scFv fusion to a Shiga toxin A Subunit (SLTA), as shown inFIGS. 2A-3B, and FIGS. 3A-3B. CD38TM4 was derived from CD38TM3 andretains the CD38 binding capacity of CD38TM3. Binding affinity of theanti-CD38 antigen binding clones in the format of scfv fusion with SLTAwas calculated and shown in Table 3.

TABLE 3 K_(D) of CD38 Binding (ng/mL) CD38-Targeting Molecule human CD38cynomolgous CD38 CD38TM2-SLTA 10.6 644 CD38TM3-SLTA 13.3 247CD38TM4-SLTA 16.4 670 CD38 targeting reference molecule 7.7 No binding#1 (CD38TR1)-SLTA

B. Example 2: Affinity Determination of Anti-CD38 Binding Clones

Cell Binding Affinity Assays were performed to quantitate the bindingaffinity of anti-CD38 binding clones to CD38-expressing cells. MOLP-8cells expressing human CD38 were suspended in 1% FBS/PBS at a viablecell concentration of approximately 2 million cells/mL. Anti-CD38binding clones formatted into IgG1 were serially diluted (2-folddilutions from 102 nM to 24.95 pM) across wells over a 6-well plate inPBS. The last well of each titration contained PBS only. Cellsuspensions and additional PBS were then added to each well so that eachwell contained a final volume of 300 μL and approximately 100,000 cells.The plate was placed into a plate shaker for 5 hours at 4° C., afterwhich the cells in each well were washed 3 times at 4° C. with PBS. 200μl of 99 nM Cγ5 goat anti-human IgG Fc specific polyclonal antibody(Jackson ImmunoResearch Laboratories, #109-175-008) was then added toeach well, and the plate was shaken for 30 minutes at 4° C. The cellswere washed again twice at 4° C. with PBS, and then analyzed byFACSCanto™ II HTS flow cytometer. Mean fluorescence intensity (MFI) of5000 events was measured for each well containing a unique antibodyconcentration, and a plot of the MFI as a function of the antibodyconcentration was fit nonlinearly with Scientist 3.0 software using theequation below to estimate the antigen binding affinity K_(D):F=p[(K_(D)+LT+n(M))−{(K_(D)+LT+n(M))2−4n(M)(LT)}^(1/2)]/2+B

where F is MFI, LT is total antibody binding site concentrationequivalent to 2 fold of antibody molecular concentration, p isproportionality constant that relates arbitrary fluorescence units tobound antibody, M is cellular concentration in molarity and is given0.553 fM based on 100,000 cells in 300 μl, n is number of receptors percell, B is background signal, and K_(D) is equilibrium dissociationconstant.

For each antibody titration curve, an estimate for K_(D) was obtained asP, n, B, and KD were floated freely in the nonlinear analysis. For adetailed derivation of the above equation, see Drake and Klakamp (2007),“A rigorous multiple independent binding site model for determiningcell-based equilibrium dissociation constants,” J. Immunol. Methods 318:157-62, which is incorporated by reference herein. Table 4 lists themeasured K_(D)s for anti-CD38 antibody clones. The antibody binding siteconcentration (2× molecular concentration) was used for the nonlinearcurve-fitting.

TABLE 4 Clone K_(D) (M) CD38TM3 1.9E−09 CD38TM1 3.2E−08 CD38TM2 3.4E−08CD38TM5 3.4E−08 CD38TM6 4.6E−09

C. Epitope Mapping of Anti-CD38 Antigen Binding Clones

In order to identify the contact residues on the CD38 extracellulardomain (ECD) by anti-CD38 antigen binding clones, epitopes mapping wascarried out by Integral Molecular. Epitopes were mapped by shotgunmutagenesis, a standard assay setup with alanine scanning and additionalmutations to change the amino acids from the human to cynomolgus residueas appropriate. Briefly, wildtype or mutated human CD38 ECD wasexpressed on the cell surface of HEK293 cells, and then the ability ofan anti-CD38 antigen binding clone of interest to bind to the CD38 ECDwhen certain amino acids were mutated was compared to the binding of thesame anti-CD38 antigen binding clone to wildtype CD38 ECD by flowcytometry. The assay was controlled for mutations that affect expressionor general folding of the molecule with a series of control moleculesthat also target CD38. Table 5 shows the binding to a surface expressedCD38 ECD protein with a specific mutation as a percentage of binding towildtype CD38. Values marked in bold indicate the amino acids that arecritical for binding and surface accessibility (based on published CD38structure) of the anti-CD38 antigen binding clones.

TABLE 5 Human CD38 ECD CD38TM1 CD38TM2 CD38TM4 CD38TM3 CD38TR1Daratumamab Amino Mean % Mean % Mean % Mean % Mean % Mean % Acid of ofof of of of Position Mutation wildtype wildtype wildtype wildtypewildtype wildtype 78 R78A  2%  3% 106%  130%  80% 52% 81 D81A  8%  9%109%  143%  128%  292%  119 C119A 108%  108% 12% 26% 83% 85% 124 L124A84%  92% 23% 43% 259%  169%  201 C201A 90%  58%  4%  5% 130%  109%  216F216A 89%  74% 18% 29% 25% 140%  230 L230A 72% 111%  7% 19% 17% 64% 254C254A 48%  81%  7% 38% 46% 67% 262 L262A 68%  97% 12% 32% 33% 83% 272Q272A 145%  135% 68% 119%  191%  167%  272 Q272R 105%  101% 71% 106% 14% 162%  273 F273A 157%  117% 89% 135%  10% 197%  274 S274F 93% 134%101%  89% 155%  27% 274 S274F 150%  124% 80% 139%  74% 14% 275 C275A 18% 37% 0.3%  17% 14% 18%

Anti-CD38 antigen binding clones CD38TM1 and CD38TM2 share the samecritical contact residues (R78 and D81) on human CD38. Critical residuesfor CD38TM5 on human CD38 include R78, H79 and D81 (data not shown).Critical residues for CD38TM3 on human CD38 include F216, L262, L124,C201, L230, C254, and S275. Critical residues for CD38TM4 on human CD38include F216, L262, C201, L230, C254, and S275. FIG. 4 shows thelocation of critical residues for CD38TM3, including surface accessiblecontact residues F216, L262 shown in red; critical structural residuesL124, C230 shown in purple, and critical structural residuesparticipating in disulfide pairs C119/C201 and LC254/C275 shown in grey.

CD38TM1, CD38TM2 and CD38TM5 bind non-overlapping epitope residues onhuman CD38 compared with daratumumab. CD38TM6 binds overlapping epitoperesidues on human CD38 with daratumumab. CD38TM3 and CD38TM4 bind anepitope partially overlapping with the CD38TR1 and daratumumab on humanCD38. CD38TR1 binds to the epitope including F216, L262, Q272, and F273on human CD38. Daratumumab binds to the epitope including F274 and F275on human CD38.

Clone CD38TM4 and/or CD38TM3 bind an epitope partially overlapping withthe reference molecule CD38TR1 and probably daratumumab on human CD38.FIG. 4 shows the location of critical residues for CD38TM4 on human CD38(surface accessible contact residues F216, L262 shown in red; criticalstructural residues L124, C201 shown in purple, and critical structuralresidues participating in disulfide pairs C119/C201 and LC254/C275 shownin grey).

Binding of anti-CD38 antigen binding clones to CD38 in the presence ofexisting CD38 antibodies including daratumumab, HB-7 (Santa Cruz, CatNo: sc-18858), AT13-5 (Thermo Fisher, Cat No: MA5-16578) and OKT-10(Sigma, Cat No: 87021903) were tested. CD38TM2, CD38TM1 and CD38TM3 wereformatted into scFv and fused to Shiga toxin A subunit as describedabove. MOLP-8 cells were incubated with 20 μg/ml of an existinganti-CD38 antibody (˜130 nM) or the control molecule (Shiga toxin Asubunit alone) for 70 minutes. Then 0.5 Wml of an anti-CD38/Shiga toxinfusion molecule (˜5 nM) was added and incubated with the cells for 48hours. Antibody binding to CD38 was detected using a primary antibodyrecognizing Shiga toxin A subunit, and a FITC conjugated anti-IgGsecondary antibody followed by flow cytometry. Mean fluorescenceintensity (MFI) of anti-CD38 antigen binding clones was calculated aftersubtracting the signal from the secondary antibody only control.

As shown in FIGS. 5A and 5B, CD38TM1 and CD38TM2 in the scFv SLTA fusionprotein format are able to bind to CD38 in the presence of daratumumab,HB-7, AT13-5, and OKT-10, indicating that they bind to an epitope onCD38 different from the epitope(s) bound by daratumumab, HB-7, AT13-5,and OKT-10. CD38TM3 in the scFv SLTA fusion protein format is able tobind to CD38 in the presence of OKT-10, but not daratumumab, HB-7 andAT13-5, indicating that CD38TM3 binds to an epitope on CD38 differentfrom the epitope bound by OKT-10, and CD38TM3 shares or partially sharesan epitope on CD38 with daratumumab, HB-7 and AT13-5.

Competitive binding among anti-CD38 antigen binding clones to CD38 wasalso tested. CD38TM2, CD38TM1 and CD38TM3 were formatted into scFv andfused to Shiga toxin A subunit as described above. MOLP-8 cells wereincubated with 20 μg/ml of an anti-CD38 binding clone (˜400 nM) or thecontrol molecule (Shiga toxin A subunit alone) for 70 minutes. Then 0.5μg/ml of an anti-CD38 antigen binding clone in the scFv SLTA fusionprotein format was added and incubated with the cells for 48 hours. CD38binding was detected by a primary antibody recognizing Shiga toxin Asubunit, and a FITC conjugated anti-IgG secondary antibody followed byflow cytometry. Mean fluorescence intensity (MFI) of anti-CD38 antigenbinding clones was calculated after subtracting the signal from thesecondary antibody only control, and plotted as a percentage of MFImeasured from Shiga toxin A subunit alone as a control. As shown inFIGS. 5C and 5D, CD38TM1 and CD38TM2 bind to CD38 in a competitivemanner, consistent with the fact that they bind to the same epitope onCD38. CD38TM3 binds to CD38 in a semi-competitively manner with CD38TM1and CD38TM2.

D. Induction of CDC and ADCC by Anti-CD38 Antibody

Anti-CD38 binding clones were formatted as human IgG1 and tested fortheir ability to induce complement-dependent cytotoxicity (CDC) andantibody-dependent cell-mediated cytotoxicity (ADCC).

For CDC assay, MOLP-8 cells were plated at a density of 10,000 cells perwell in a black 96-well flat-bottom tissue culture plate in 50 μL ofcomplete media (RPMI supplemented with 10% fetal bovine serum). 50 μL ofanti-CD38 antibody (9 μg/ml), control IgG1 antibody (9 μg/ml), or mediaalone was added to each well and left to incubate at room temperature(RT) for 10 min. Varying amounts (2-15 μL) of purified rabbit complement(Cat # CL 3441 Cedarlane Laboratories, Canada) was added to each wellexcept control wells. After one-hour incubation at 37° C., the plate wasbrought to room temperature, 100 μL of cell titer CytoTox Glo™ reagent(Promega G7571/G7573) was added to each well, the plate was shaken for 5to 7 minutes, and luminescence was read on an EnVision® (Perkin Elmer)luminescence plate reader. The conditions tested were: cells alone;cells+complement; cells+IgG1 control+complement;cells+antibody+complement. % CDC was calculated using the followingequation: 100−(RLUT/RLUC)×100), where RLUT is the relative luminescenceunits of the test sample and RLUc is the relative luminescence units ofthe sample with complement alone. The assay was conducted intriplicates, and statistical analysis was performed using PRISMsoftware.

For measuring ADCC, MOLP-8 cells were used as target cells. Peripheralblood mononuclear cells (PBMCs) were isolated as effector cells byFicoll-Plaque™ separation from buffy coat or LRS which were obtainedfrom the Stanford Blood Center (Palo Alto, Calif.). Specimens werediluted 1:3 with 2% FBS in PBS. 15 mL of Ficoll-Plaque™ (GE Healthcare)was gently layered under 35 mL of diluted specimen and centrifuged at1800 rpm (brake off) for 25 minutes. The cloudy interphase containingPBMCs was collected, washed 3 times in 2% FBS/PBS and frozen intoaliquots of 50×10⁶ cells/mL per aliquot in 10% DMSO/FBS. Before use,frozen aliquots of PBMCs were thawed and cultured overnight in 10%FBS/RPMI+5 ng/mL recombinant human IL2 (R & D systems #202-IL) at 2×10⁶cells per mL.

For the ADCC assay, all steps were performed in complete media. 5000target cells were plated per well in a 96-well plate, and 50 μL ofanti-CD38 antibody (10 μg/ml), control IgG1 (10 μg/ml), or media alonewas added to each well. 50 μL of human effector PBMCs was then added tothe wells at a ratio of between 1:25 to 1:50 for target:effector (T:E)cells. The plate was briefly centrifuged for 30 seconds at 800 rpm tobring all cells into close proximity. After 4 hours of incubation at 37°C., the plate was centrifuged at 1100 rpm for 5 minutes and 100 μLsupernatant was transferred to a white plate. 100 μL CytoTox Glo™reagent (Promega cat # G9292) was added to the supernatant and the platewas then shaked for 20-30 minutes at RT. Luminescence was read on anEnVision® (Perkin Elmer) luminescence plate reader and percentage ofspecific lysis was calculated using the following equation:(RLUT/RLUE/T)/(RLUL/RLUE/T)×100, where RLUT is the relative luminescenceunits of the test sample and RLUE/T is the relative luminescence unitsof the sample containing target cells and effector cells alone, and RLULis the relative luminescence units for cells lysed with Triton X-100.The assay was conducted in triplicates, and statistical analysis wasperformed using PRISM software.

The percentages of CDC and ADCC induced by anti-CD38 antibody clones areshown in Table 6. All anti-CD38 antibody clones are capable of inducingCDC and ADCC towards CD38 expressing cells.

TABLE 6 % CDC % ADCC Clones (mean) (mean) CD38TM3 16.3 49.2 CD38TM1 15.029.0 CD38TM2 7.2 31.8 CD38TM5 4.7 35.2 CD38TM6 34.3 50.4

E. Derivation of CD38TM4 from CD38TM3

VH and VL of CD38TM3 do not bind to standard monoclonal antibodypurification resins (protein A or protein L), and it was purified usingthe His-tag and IMAC column (FIG. 11A showing purification of CD38TM3 inthe scFv-SLTA fusion protein format as an example). In order to rendereasier purification, the light chain of CD38TM3 which is a Lambda lightchain was modified by framework mutations so that it is able to bind toProtein L, allowing for affinity purification without an additional tag.As show in FIG. 11C, the first 21 amino acids of CD38TM3 VL domain wasreplaced with the first 22 amino acids of the VL domain of CD38TR1,which is a Kappa light chain to derive the VL domain of CD38TM4. Theresulting CD38TM4 is able to bind to protein L and thus was purified byProtein L column (FIG. 11B showing purification of CD38TM4 in thescFv-SLTA fusion protein format as an example).

1.-27. (canceled)
 28. A composition comprising an anti-CD38 antigen binding domain comprising: a) a variable heavy domain (VH) comprising: i) a VHCDR1 comprising the sequence of SEQ ID NO:2; ii) a VHCDR2 comprising the sequence of SEQ ID NO:3; and iii) a VHCDR3 comprising the sequence of SEQ ID NO:4; and b) a variable light domain (VL) comprising: i) a VLCDR1 comprising the sequence of SEQ ID NO:6; ii) a VLCDR2 comprising the sequence of SEQ ID NO:7; and iii) a VLCDR3 comprising the sequence of SEQ ID NO:8.
 29. A composition according to claim 28 wherein said VH comprises the sequence of SEQ ID NO:1 and said VL comprises the sequence of SEQ ID NO:25.
 30. A composition according to claim 28 wherein said VH comprises the sequence of SEQ ID NO:1 and said VL comprises the sequence of SEQ ID NO:5.
 31. A composition according to claim 28 wherein said VH and said VL are in a single polypeptide.
 32. A composition according to claim 31 wherein said polypeptide comprises a scFv linker and said polypeptide has the orientation from N- to C-terminal of VH-scFv linker-VL.
 33. A composition according to claim 31 wherein said polypeptide comprises a scFv linker and said polypeptide has the orientation from N- to C-terminal of VL-scFv linker-VH.
 34. A composition according to claim 28 wherein the composition comprises a first polypeptide comprising said VH and a second polypeptide comprising said VL.
 35. A composition according to claim 28 wherein said composition is an antibody comprising: a) a heavy chain comprising said VH; and b) a light chain comprising said VL.
 36. A composition according to claim 35 wherein said heavy chain comprises said variable heavy domain and a heavy constant domain selected from the group consisting of the heavy constant domains of human IgG1, IgG2 and IgG4, or variants thereof.
 37. A composition according to claim 36 wherein said heavy constant domain is the heavy constant domain of human IgG1.
 38. A composition according to claim 36 wherein said heavy constant domain is a variant of the heavy constant domain of human IgG1.
 39. A composition according to claim 38 wherein said variant heavy constant domain of human IgG1 has ablated FcγR binding.
 40. A composition according to claim 36 wherein said heavy constant domain is a variant of the heavy constant domain of human IgG4 comprising a S228P amino acid substitution compared to the wild-type heavy constant domain of human IgG4.
 41. A nucleic acid composition encoding said composition according to claim 34, wherein said nucleic acid composition comprises: a) a first nucleic acid encoding said first polypeptide; and b) a second nucleic acid encoding said second polypeptide.
 42. An expression vector composition comprising: a) a first expression vector comprising said first nucleic acid of claim 41; and b) a second expression vector comprising said second nucleic acid of claim
 41. 43. A host cell comprising said expression vector composition according to claim
 42. 44. A method of making a composition comprising an anti-CD38 antigen binding domain, comprising culturing said host cell of claim 43 under conditions wherein said composition comprising the anti-CD38 binding domain is expressed, and recovering said composition.
 45. A method of treating multiple myeloma comprising administering to a subject in need thereof an effective amount of said composition according to claim
 28. 46. A composition comprising an anti-CD38 antigen binding domain comprising: a) a variable heavy domain (VH) comprising: i) a VHCDR1 comprising the sequence of SEQ ID NO:10; ii) a VHCDR2 comprising the sequence of SEQ ID NO:11; and iii) a VHCDR3 comprising the sequence of SEQ ID NO:12; and b) a variable light domain (VL) comprising: i) a VLCDR1 comprising the sequence of SEQ ID NO:14; ii) a VLCDR2 comprising the sequence of SEQ ID NO:15; and iii) a VLCDR3 comprising the sequence of SEQ ID NO:16.
 47. A composition comprising an anti-CD38 antigen binding domain comprising: a) a variable heavy domain (VH) comprising: i) a VHCDR1 comprising the sequence of SEQ ID NO:18; ii) a VHCDR2 comprising the sequence of SEQ ID NO:19; and iii) a VHCDR3 comprising the sequence of SEQ ID NO:20; and b) a variable light domain (VL) comprising: i) a VLCDR1 comprising the sequence of SEQ ID NO:22; ii) a VLCDR2 comprising the sequence of SEQ ID NO:23; and iii) a VLCDR3 comprising the sequence of SEQ ID NO:24.
 48. A composition comprising an anti-CD38 antigen binding domain comprising: a) a variable heavy domain (VH) comprising: i) a VHCDR1 comprising the sequence of SEQ ID NO:54; ii) a VHCDR2 comprising the sequence of SEQ ID NO:55; and iii) a VHCDR3 comprising the sequence of SEQ ID NO:56; and b) a variable light domain (VL) comprising: i) a VLCDR1 comprising the sequence of SEQ ID NO:58; ii) a VLCDR2 comprising the sequence of SEQ ID NO:59; and iii) a VLCDR3 comprising the sequence of SEQ ID NO:60. 